ir

Available online at www.sciencedirect.com

ctExosomesAmong the many vesicles and macromolecules secretedby cells are nanometer-sized bodies known as exosomes.These were first described in the 1980s as nanovesiclesreleased by exocytosis from reticulocytes [2]. These smallvesicles are released by most cells in culture and a rangeof cell types in vivo including B cells, dendritic and mast

multiple anatomic sites often in a targeted fashion whosemechanism remains unknown.

Exosomes have several features that make them usefulfor biomedical purposes. Since many body fluids containexosomes encapsulating miRNA, they may be useful asearly biomarkers of disease [16]. These may be particu-larly useful for diagnosis and prognostication of not only

www.sciencedirect.com Current Opinion in Biotechnology 2014, 28:6974Biological nanoparticles and theSarah Stanley

Over millions of years, biological systems have evolved wide

varieties of nanoparticles. Naturally occurring nanoparticles

show great diversity: they may be intracellular or extracellular,

formed of organic or inorganic materials and have wide-ranging

biological roles. Despite this diversity, nanoparticles found in

nature possess several characteristics that make them

attractive for biomedical purposes. This review presents an

overview of the most common biological nanoparticles and

outlines the potential applications of natural and modified

biological nanoparticles.

Addresses

Laboratory of Molecular Genetics, Rockefeller University, New York, NY

10065, USA

Corresponding author: Stanley, Sarah (sstanley@mail.rockefeller.edu)

Current Opinion in Biotechnology 2014, 28:6974

This review comes from a themed issue on Nanobiotechnology

Edited by Jonathan S Dordick and Kelvin H Lee

For a complete overview see the Issue and the Editorial

Available online 8th January 2014

0958-1669/$ see front matter, # 2013 Elsevier Ltd. All rightsreserved.

http://dx.doi.org/10.1016/j.copbio.2013.11.014

IntroductionNanotechnology research has primarily focused on man-made particles, but naturally occurring nanoparticles havebeen present for millions of years [1]. A naturally occur-ring nanoparticle is an assembly of molecules or atoms,synthesized in a biological system, with at least onedimension in the 1100 nm range. These particles in-clude intracellular structures such as magnetosomes andextracellular assemblies such as lipoproteins and viruses.Their functions are diverse, ranging from mineral storagedepots to intercellular communication. The aims of thisreview were twofold: first, to give an overview of nano-particles that occur in biological systems, their formationand biological function and second, to describe how thesebiological nanoparticles are being modified and used forbiomedical applications.

ScienceDire influence on organisms

cells, neurons [3], adipocytes [4], mesenchymal stem cells[5] and endothelial cells, in addition to tumour cells [6].They are also released into many body fluids such asplasma, urine, saliva, cerebrospinal fluid, breast milk, andamniotic and ascitic fluid [7].

Exosomes are formed by budding of the endosomal mem-brane into the lumen to form multivesicular bodies whichare then released at the cell surface [8]. Exosomes arecharacterized by a lipid bilayer outer surface, by their sizeof approximately 50160 nm and by their density (1.131.19 g/ml) [9]. Exosome membranes have a unique com-position with some highly conserved proteins common tomany exosomes and others that depend on the type andstate of the cell of origin. The exosomal membranesalso have a high content of cholesterol and sphingolipidsassociated with lipid rafts, ceramide and phosphoglycer-ides with long and saturated fatty-acyl chains [10] inaddition to a number of saccharide groups [11].

Exosomes encapsulate significant numbers of proteinsand nucleic acids. On the basis of their size, it is estimatedthat exosomes may transport up to 100 proteins and10 000 nucleotides. Exosomes have been demonstratedto contain mRNA, miRNA and other non-coding RNAs.Much of these are in short fragments (

malignancy [6] but also diseases such as Alzheimersdisease [17]. The role of exosomes in antigen presen-tation also makes them valuable in vaccination, both forinfectious diseases and for tumours. In particular, theypresent antigens for long periods and are very stable.Current trials include using autologous dendritic cell-derived exosomes to which a tumour antigen has beenattached for use as a tumour vaccine for small cell lungcarcinoma and melanoma [18]. Finally, they may also

which are smaller and denser. These particles transportendogenous TAG, cholesterol esters and fat-soluble vita-mins from the liver to peripheral tissues with apolipopro-tein B-100. In the periphery, TAG is hydrolysed bylipoprotein lipase found on the surface of vascular endo-thelial cells. Removal of TAG, with protein and phos-pholipid transfer to high density lipoproteins (HDL),converts VLDL into smaller, denser low density lipopro-teins (LDL) [23]. LDL are composed of cholesterol andcholesterol esters along with ApoB-100 and transport

70 Nanobiotechnology

d

t%)offer a mechanism for targeted protein or nucleic aciddelivery. The exosome contents are protected fromdegradation and are not endocytosed by macrophages,so they have a long circulating half-life, achieve a higherconcentration and are less toxic. Recent work has usedmodified dendritic cells to release exosomes with a sur-face neuron-specific protein loaded with synthetic siRNAfor targeted CNS gene knockdown [19]. Further studiesare examining the utility of using synthetic exosomes fortargeted delivery of drugs from anti-inflammatory agentssuch as curcumin to nucleic acid chelators in malignancy[20].

LipoproteinsLipoproteins are complex self-assembling structures oflipids and specialized proteins, apolipoproteins, thattransport water-insoluble lipids in the aqueous internalenvironment of vertebrates and insects. Lipids and apo-lipoproteins form coreshell spherical or discoidal nano-particles of 7 to >80 nm (see Table 1). They arecomprised of a core of non-polar lipids, triacylglyerolsand esterified cholesterol with a surface layer of apolipo-proteins, phospholipids and non-esterified cholesterol[21].

Lipoproteins are synthesized primarily by the liver andintestines and change in structure and composition astheir components are used by peripheral tissues. Lipo-proteins are defined by their size, density and proteincontent (see Table 1). The largest, least dense lipopro-teins are chylomicrons (CM). These are formed fromdietary free fatty acids and monoacylglycerols convertedto triacylglycerols (TAG) in the enterocytes. TAG is thenpackaged with dietary cholesterol and apolipoprotein B-48 [22]. Next are very low density lipoproteins (VLDL)

Table 1

Composition and physical properties of lipoproteins

Size (nm) Density (g/ml) Total lipi

content (w

Chylomicrons 200600

Biological nanoparticles and their roles in vivo Stanley 71added to the lipoprotein core using hydrophobicallycoated inorganic nanocrystals such as gold or iron oxide[29]. Modification of the lipoprotein shell allows targetedcell uptake, for example, by macrophages in atherosclero-tic plaques [30].

HDL are also proposed as therapies for infections and fordrug delivery. Unmodified HDL nanoparticles bind lipo-polysaccharide, released from gram negative bacteria, toprevent overstimulation of the immune system [31].HDL nanoparticles can also be used as decoys to preventdamage to healthy cells either by viruses or toxins thatbind to specific cell surface proteins. Further, incorporat-ing membrane components from pathogens in rHDLparticles allows their use as vaccines [32]. Other bioac-tive lipid moieties can also be incorporated into rHDLparticles. For example, incorporation of sphingosine-1-phosphate into the lipoprotein shell induces endothelialcell proliferation and tube formation that may beapplicable to acute coronary syndromes.

The lipoprotein core can also be used for therapeuticdelivery of lipophilic drugs to increase their solubilityand bioavailability and to reduce toxicity [33]. Severalcompounds have been successfully incorporated intoHDL. These include delivery of amphotericin B totreat fungal and protozoal infections in mice withoutthe toxicity normally associated with amphotericin B.Modified surface apolipoproteins may also direct thera-pies to defined cell types. With the development ofmechanisms for controlled release, reconstituted lipo-proteins may form even more valuable tools for therapydelivery [27].

FerritinIn addition to organic nanoparticles, organisms also pro-duce inorganic nanoparticles, in particular iron-containingparticles such as ferrihydrite and magnetite. Ferritin isexpressed in bacteria, archaea and eukaryotes. Its primaryfunctions are as a protein nanocage to synthesize and storeiron oxides and to sequester potentially damaging ironions [34]. In eukaryotes, ferritin is a protein complexcomposed of 24 subunits organized into a four-helicalbundle to form a hollow, symmetrical, almost sphericalprotein shell of 12 nm. The interior cavity may accom-modate up to 4500 Fe atoms. In eukaryotes, there are twomajor ferritin genes encoding heavy (H) and light (L)chains with differing properties (the L chain lacks thecatalytic activity of the H chain) that assemble to formheteropolymers. The ratio of H to L chains varies fromtissue to tissue to form a wide range of isoferritins [35].

Ferritin acts as an iron storage molecule and preventsgeneration of hydroxyl radicals by oxidation of Fe(II) to

Fe(III). In ferritin with high H chain content and catalyticactivity, highly ordered ferric oxohydroxide, is formed butin ferritin with high L chain content, the crystal structure

www.sciencedirect.com is more disordered. The magnetic properties of the ferri-tin core are complex. It is thought that the Fe(III) ions areantiferromagnetically coupled with a superparamagneticmoment [36]. Release of iron from ferritin requires elec-trons, protons and water release, although little is knownabout the process. Iron exits through channels in theferritin shell or by proteolytic degradation of ferritin,possibly in lysosomes [37].

In addition to H and L chains, mitochondrial ferritin, withan N-terminal extension and mitochondrial localizationsignal, has been identified in humans and mice. Thisprotein has ferroxidase activity and the ability to seques-ter iron. In humans, it is present in testis and spermatozoaand in mice it is also found in heart, CNS, kidney andpancreatic islets. Mitochondria produce high levels ofreactive oxygen species and also need iron for enzymes,suggesting the mitochondrial ferritin may have both ironstorage and antioxidant roles [38].

The apoferritin shell can be used as a chamber for thesynthesis of nanoparticles. Metal nanoparticles with Cr,Co, In oxides and nickel hydroxide and semiconductornanoparticles have been made in ex vivo horse spleenapoferritin. Using the apoferritin shell produces nanopar-ticles with highly reproducible shape and size. Inaddition, ferritin shells can be adsorbed in a definedarrangement either using their negative charge or bymodification of the ferritin subunit, for example, to in-clude a hydrophobic terminal to bind to carbonaeousmaterial or titanium binding peptides. The protein shellscan subsequently be removed to leave the nanoparticlesin situ [39]. Arrays of ferritin nanoparticles have been usedfor several purposes, from carbon nanotube growth tonanodisk fabrication and development of a bionanobat-tery [40].

The ferritin iron oxide core makes it an attractive candi-date for development of MR contrast agents. Endogenousferritin is detectable by MRI and can be used as a cellmarker [41]. Modified ferritin, with N,N dimethyl-1,3propanediamine (DMPA) coupled to surface proteins,was used to detect negatively charged basement mem-branes, while addition of biotinylated peptides allowstargeting to specific cells [30]. Modification of the ferritinshell to alter its catalytic activity allows the iron core to bereplaced by gadolinium for imaging, photosensitizers anddrugs for tumours, and quantum dots for imaging [42].Ferritin has also been used to transduce the effects ofradiofrequency fields into channel activation to controltransgene expression in vitro [43].

MagnetiteWhile almost all bacteria have ferritin, a specialized group

of magnetotactic bacteria have evolved an additional iron-containing nanoparticle [44]. These bacteria have aspecialized organelle, the magnetosome, comprised of a

Current Opinion in Biotechnology 2014, 28:6974

72 Nanobiotechnologylipid bilayer and containing magnetic iron-containingminerals, magnetite or greigite. Magnetosomes are 5070 nm in diameter and can form one or more chains,aligning the bacteria to the earths magnetic field andfacilitating the bacteria in swimming to more oxygen-depleted regions.

The magnetosome membrane is a lipid bilayer formed bythe invagination of the inner cell membrane. The mem-brane contains a unique set of 2040 proteins which havebeen implicated in the formation of the membrane, inregulating its size and shape and in controlling the for-mation of the magnetosome chain(s) (see [45] forreview). Iron uptake and subsequent magnetite formationoccur only when the environment is almost anaerobic.The magnetotactic bacteria employ both common irontransporters and unique transporters, such as MagA,which may encode an ATP-dependent iron transporter[46]. The mechanism for magnetite formation is not wellunderstood. Soluble cytoplasmic iron may be oxidized toa ferrihydrite precursor, then moved into the magneto-some and reduced to magnetite. Alternatively, an iron-containing ferritin-like protein and soluble iron co-pre-cipitate, forming crystals at the cell membrane that ma-ture in the magnetosome to magnetite. The initialmagnetite crystals formed are small and superparamag-netic, but once they are greater than 35 nm, they becomea stable single domain magnetic crystal [47].

Magnetite has also been detected in additional species. Inhoney bees, 7.5 nm spherical magnetite particles are foundin iron deposition vesicles of trophocytes [48]. It is alsopresent in the denticles of certain marine mollusks [49] andin the nasal region of birds such as Bobolinks and pigeons[50]. Magnetite has also been extracted from human tissue,including the hippocampus [51], and possibly also withinthe plaques of Alzheimers disease [52]. Magnetite inBobolinks and pigeons has been implicated in their abilityto use magnetic fields for navigation [53], though theevidence for the involvement of magnetite is incomplete[54]. The role of magnetite in the human brain isunknown, though it has been suggested that biogenicmagnetite may transduce magnetic signals produced byneurons and astrocytes within the neocortex [55].

Magnetite nanoparticles from magnetotactic bacteriahave a narrow size distribution, uniform morphologyand lower toxicity than those produced by chemicalsynthesis. Magnetosomes are being used for several invitro purposes including removal of heavy metals fromwater by surface adsorption, magnetic separation, immu-nobinding, receptor binding assays, and DNA extraction.In vivo, they have been studied as contrast agents for MRIand may be useful for heating applications such as tumour

hyperthermia or regulated cell activation. Several of theseapplications require functionalization of the magneto-some either by chemical coupling to lipids or proteins

Current Opinion in Biotechnology 2014, 28:6974 in the membrane or by binding to endogenous membraneproteins [56]. However, the magnetosome membrane canalso be modified by transgenic expression of monomericcamelid antibodies, resulting in genetically encodedexpression of a magnetosome surface antibody. Biogenicmagnetite itself can also be modified, for example, bycross-linking to chitosan followed by coating with silvernanoparticles for antibacterial uses or linking to trypsinfor protein digestion.

VirusesViruses, small infectious particles that require living cellsfor replication, are highly diverse, naturally occurringnanoparticles. They share a common structure of a shellcomprised of capsid proteins enclosing the DNA or RNAviral genome. They span a wide variety of sizes (withinthe nanometer range) and morphologies from simplespheres to rods to icosahedrons. Viruses have beendescribed that target almost all known organisms andtissues. Most applications use virus-like particles (VLP)which are native viral capsid proteins without nucleic acidand therefore do not cause infection [57].

Viruses and VLP have defined geometries, are veryuniform and have robust protein shells that can be modi-fied for bioconjugation or for chemical modification,allowing molecules to be displayed in a precise spatialdistribution. However, they can be difficult to produce inbulk and there are limits to the size of antigen that can beattached. In addition, virus capsid protein folding is notalways well understood. Bioconjugation to capsid lysineor cysteine residues is relatively straightforward andallows extensive attachment of molecules. The capsidsare stable over a wide range of temperatures and pH,making them suitable for many applications.

Several in vitro applications of VLP have been describedincluding use as nanoreactors and as filamentous orspherical scaffolds [42]. Modified VLP are also beingused in vivo. Their primary use has been in vaccinationeither to induce immunity against the parent virus or tomodify other diseases [58]. VLP conjugated to appropri-ate epitopes have been used for anti-tumour vaccines andfor vaccines against chronic diseases such as hyperten-sion. VLP have also been modified for use as contrastagents in MRI, for example, by addition of gadolinium tometal binding sites or conjugation of gadolinium chelates,while VLP labelled with unstable isotopes of fluoridehave been used as PET contrast agents [30]. VLP canalso be modified to create hydrophobic pockets within theprotein shell which can be loaded with insoluble, hydro-phobic drugs for delivery [59]. In addition, the naturalaffinity of VLP for defined cell types allows targeted

delivery or the VLP can be modified by bioconjugationto targeting molecules, such as folic acid, for cell-specificdelivery.

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Biological nanoparticles and their roles in vivo Stanley 73ConclusionOver the last few years, there have been many studiesaimed at increasing our understanding of the structure,synthesis and physiological roles of naturally occurringnanoparticles. Biologically produced nanoparticles arehighly diverse but also offer features that make themattractive for biomedical uses: the uniformity of theirstructure, low toxicity, ability to evade the immunesystem and capacity for modification. These propertiesare now beginning to be harnessed both for in vitrosystems such as diagnostics and for in vivo purposes. Asour knowledge of the biology of naturally occurringnanoparticles expands and challenges related to synthesisof such particles are overcome, it is likely that the bio-medical applications of natural nanoparticles will expandfurther.

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

of special interest of outstanding interest

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Biological nanoparticles and their influence on organismsIntroductionExosomesLipoproteinsFerritinMagnetiteVirusesConclusionReferences and recommended reading